Live cell imaging of protein trafficking: Dynamics and functional insights

Mehta, Kritika

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https://hdl.handle.net/2142/125773 Copy

Description

Title

Live cell imaging of protein trafficking: Dynamics and functional insights

Author(s)

Mehta, Kritika

Issue Date

2024-07-10

Director of Research (if dissertation) or Advisor (if thesis)

Zhang, Kai

Doctoral Committee Chair(s)

Zhang, Kai

Committee Member(s)

• Fratti, Rutillo A
• Tsai, Nien-Pe
• Stadtmueller, Beth M

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

activity-regulated cytoskeleton-associated protein, phospholipids, virus-like capsid, fluorescence imaging, super resolution imaging, Minflux

Abstract

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene that plays a vital role in learning and memory. Arc protein has structural and functional properties similar to viral Group-specific antigen (Gag) protein and mediates the intercellular RNA transfer through virus-like capsids. However, the regulators and secretion pathway through which Arc capsids maneuver cargos are unclear. Here, we identified that phosphatidylinositol-3-phosphate (PI3P) mediates Arc capsid assembly and secretion through the endosomal-multivesicular body (MVB) pathway. Indeed, reconstituted Arc protein preferably binds to PI3P. In HEK293T cells, Arc forms puncta that colocalizes with FYVE, an endosomal PI3P marker, as well as Rab5 and CD63, early endosomal and MVB markers, respectively. Super-resolution imaging resolves Arc accumulates within the intraluminal vesicles of MVB. CRISPR double knockout of RalA and RalB, crucial GTPases for MVB biogenesis and exocytosis, severely reduces the Arc-mediated RNA transfer efficiency. RalA/B double knockdown in cultured rat cortical neurons increases the percentage of mature dendritic spines. Intake of extracellular vesicles purified from Arc-expressing wild-type, but not RalA/B double knock-down, cells in mouse cortical neurons reduce their surface GluA1 level. These results suggest that, unlike the Human Immunodeficiency Virus Gag, whose membrane targeting requires interaction with plasma-membrane-specific phosphatidyl inositol (4,5) bisphosphate (PI(4,5)P2), Arc capsids assemble at the endocytic membranes is mediated by PI3P. Understanding Arc’s secretion pathway helps gain insights into its role in intercellular cargo transfer and highlights the commonality and distinction of trafficking mechanisms between structurally resembled capsid proteins.

Graduation Semester

2024-08

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/125773

Copyright and License Information

© 2024 Kritika Mehta

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